RESUMEN
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD. Analysis of homozygous mutant embryos from 18 litters yielded 25% with normal organ situs, 35% with situs inversus totalis, and 40% with heterotaxy. Embryos with heterotaxy had complex structural heart defects that included discordant atrioventricular and ventricular outflow situs and atrial/pulmonary isomerisms. Variable combinations of a distinct set of cardiovascular anomalies were observed, including superior-inferior ventricles, great artery alignment defects, and interrupted inferior vena cava with azygos continuation. The surprisingly high incidence of heterotaxy led us to evaluate the diagnosis of PCD. PCD was confirmed by EM, which revealed missing outer dynein arms in the respiratory cilia. Ciliary dyskinesia was observed by videomicroscopy. These findings show that Dnahc5 is required for the specification of left-right asymmetry and suggest that the PCD-causing Dnahc5 mutation may also be associated with heterotaxy.
Asunto(s)
Trastornos de la Motilidad Ciliar/patología , Dineínas/genética , Cardiopatías Congénitas/ultraestructura , Mutación , Situs Inversus/ultraestructura , Animales , Cilios/genética , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/fisiopatología , Modelos Animales de Enfermedad , Genes Recesivos , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Humanos , Pulmón/fisiopatología , Pulmón/ultraestructura , Ratones , Ratones Mutantes , Miocardio/ultraestructura , Situs Inversus/genética , Situs Inversus/fisiopatología , Vena Cava Inferior/fisiopatología , Vena Cava Inferior/ultraestructuraRESUMEN
Human induced pluripotent stem cells (iPSCs) that express stable and robust fluorescent proteins have proven to be indispensable in basic and translational research. These reporter iPSC lines can greatly facilitate cell imaging, sorting, and tracking in vitro and in vivo studies. Here, we document two reporter human iPSC lines generated by gene-editing technologies that precisely integrated one-copy of a tdTomato transgene driven by strong CAG promoter into the AAVS1 human safe harbor locus.
Asunto(s)
Edición Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Humanos , TransfecciónRESUMEN
Expanded human skin fibroblast cells from four different aged healthy individuals, 11-year-old female, 1-year-old male, 2-month-old male, and 8-year-old male, were used to generate integration-free induced pluripotent stem cell (iPSC) lines TRNDi021-C, TRNDi023-D, TRNDi024-D, and TRNDi025-A, respectively, by exogenous expression of four reprogramming factors, human SXO2, OCT3/4, C-MYC, KLF4. The authenticity of established iPSC lines was confirmed by the expressions of stem cell markers, karyotype analysis, and teratoma formation. These iPSC lines could serve as young healthy controls for the studies involving patient-specific iPSCs.
Asunto(s)
Células Madre Pluripotentes Inducidas , Teratoma , Anciano , Diferenciación Celular , Reprogramación Celular , Niño , Femenino , Fibroblastos , Humanos , Lactante , Cariotipo , Cariotipificación , Factor 4 Similar a Kruppel , MasculinoRESUMEN
Human-derived induced pluripotent stem cells (iPSCs) have proven to be indispensable in cardiovascular drug development, disease modeling, and developmental biology research. For this reason, it is particularly useful to develop wild-type iPSC lines to be used in experimental or control conditions. Here, we present two such cell lines generated from a sample of peripheral blood mononuclear cells (PBMCs) from a healthy patient with normal cardiac function.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular/citología , Voluntarios Sanos , Corazón/fisiología , Células Madre Pluripotentes Inducidas/citología , Población Blanca , Femenino , Humanos , Persona de Mediana EdadRESUMEN
The mechanisms regulating translation and splicing are not well understood. We provide insight into a new regulator of translation, OGFOD1 (2-oxoglutarate and iron dependent oxygenase domain-containing protein 1), which is a prolyl-hydroxylase that catalyzes the posttranslational hydroxylation of Pro-62 in the small ribosomal protein S23. We show that deletion of OGFOD1 in an in vitro model of human cardiomyocytes decreases translation of specific proteins (e.g., RNA-binding proteins) and alters splicing. RNA sequencing showed poor correlation between changes in mRNA and protein synthesis, suggesting that posttranscriptional regulation was the primary cause for the observed differences. We found that loss of OGFOD1 and the resultant alterations in protein translation modulates the cardiac proteome, shifting it towards higher protein amounts of sarcomeric proteins such as cardiac troponins, titin and cardiac myosin binding protein C. Furthermore, we found a decrease of OGFOD1 during cardiomyocyte differentiation. These results suggest that loss of OGFOD1 modulates protein translation and splicing, thereby leading to alterations in the cardiac proteome and highlight the role of altered translation and splicing in regulating the proteome..
Asunto(s)
Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Prolil Hidroxilasas/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular , Conectina , Técnicas de Inactivación de Genes , Humanos , Proteínas Nucleares/genética , Prolil Hidroxilasas/genética , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Transcriptoma , TroponinaRESUMEN
Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin-free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment with heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined Dulbecco's modified Eagle's medium/F-12-based medium on either Matrigel or defined matrices like vitronectin and Synthemax. One of heparin's main functions was to act as a Wnt modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost-effective method for cardiomyocyte derivation from hPSCs that can be used for potential large-scale drug screening, disease modeling, and future cellular therapies. Stem Cells Translational Medicine 2017;6:527-538.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medios de Cultivo/química , Heparina/farmacología , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Fenotipo , Factores de Tiempo , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
The use of mouse models is rapidly expanding in biomedical research. This has dictated the need for the rapid genotyping of mutant mouse colonies for more efficient utilization of animal holding space. We have established a high-throughput protocol for mouse genotyping using two robotics workstations: a liquid-handling robot to assemble PCR and a microfluidics electrophoresis robot for PCR product analysis. This dual-robotics setup incurs lower start-up costs than a fully automated system while still minimizing human intervention. Essential to this automation scheme is the construction of a database containing customized scripts for programming the robotics workstations. Using these scripts and the robotics systems, multiple combinations of genotyping reactions can be assembled simultaneously, allowing even complex genotyping data to be generated rapidly with consistency and accuracy. A detailed protocol, database, scripts, and additional background information are available at http://dir.nhlbi.nih.gov/labs/ldb-chd/autogene/.
Asunto(s)
Mapeo Cromosómico/métodos , Electroforesis/métodos , Ratones/genética , Técnicas Analíticas Microfluídicas/métodos , Reacción en Cadena de la Polimerasa/métodos , Robótica/métodos , Análisis de Secuencia de ADN/métodos , Algoritmos , Animales , Electroforesis/instrumentación , Genotipo , Técnicas Analíticas Microfluídicas/instrumentación , Reacción en Cadena de la Polimerasa/instrumentación , Reproducibilidad de los Resultados , Robótica/instrumentación , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/instrumentación , Programas InformáticosRESUMEN
Factors limiting the adoption of iPSC technology include the cost of developing lines and the time period that it takes to characterize and bank them, particularly when integration free, feeder free, and Xeno-free components are used. In this manuscript we describe our optimization procedure that enables a single technician to make 20-40 lines at a time in a 24-96 well format in a reliable and reproducible fashion. Improvements spanned the entire workflow and included using RNA virus, reducing cytotoxicity of reagents, developing improved transfection and freezing efficiencies, modifying the manual colony picking steps, enhancing passaging efficiency and developing early criteria of success. These modifications allowed us to make more than two hundred well-characterized lines per year.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Técnicas de Reprogramación Celular/métodos , Reprogramación Celular , Medios de Cultivo/química , Células Madre Pluripotentes Inducidas , Técnicas de Cultivo de Célula/economía , Línea Celular , Técnicas de Reprogramación Celular/economía , HumanosRESUMEN
BACKGROUND: GJA1 gene encodes a gap junction protein known as connexin 43 (Cx43). Cx43 is abundantly expressed in the ventricular myocardium and in cardiac neural crest cells. Cx43 is proposed to play an important role in human congenital heart disease, as GJA1 knock-out mice die neonatally from outflow tract obstruction. In addition, patients with visceroatrial heterotaxia or hypoplastic left heart syndrome were reported to have point mutations in GJA1 at residues that affect protein kinase phosphorylation and gating of the gap junction channel. However, as these clinical findings were not replicated in subsequent studies, the question remains about the contribution of GJA1 mutations in human congenital heart disease (CHD). MATERIALS AND METHODS: We analyzed the GJA1 coding sequence in 300 patients with CHD from two clinical centers, focusing on outflow tract anomalies. This included 152 with Tetralogy of Fallot from over 200 patients exhibiting outflow tract anomalies, as well as other structural heart defects including atrioventricular septal defects and other valvar anomalies. Our sequencing analysis revealed only two silent nucleotide substitutions in 8 patients. To further assess the possible role of Cx43 in CHD, we also generated two knock-in mouse models with point mutations at serine residues subject to protein kinase C or casein kinase phosphorylation, sites that are known to regulate gating and trafficking of Cx43, respectively. RESULTS: Both heterozygous and homozygous knock-in mice were long term viable and did not exhibit overt CHD. CONCLUSION: The combined clinical and knock-in mouse mutant studies indicate GJA1 mutation is not likely a major contributor to CHD, especially those involving outflow tract anomalies.
RESUMEN
We suggest that characterization of processes involved in differentiation of the pluripotential cardiac precursor cells in their embryonic environment will permit identifying pathways important for induction of diverse stem cells toward the cardiac phenotype. Phenotypic characteristics of cardiac cells are their contractile and electrical properties. The objective of the present study was to define whether calcium (Ca(++)) has a regulatory role in the pluripotential precursor cell population during commitment into cardiomyocytes. We used the chick embryo model because of ease of staging the embryos and visibility of heart development. Using the Ca(++) indicator Fluo-3/acetoxymethyl and confocal microscopy, we demonstrated the existence of higher free Ca(++) levels in the cardiogenic precursor cells than in neighboring cell populations outside of the heart fields. Subsequently, gastrulation stage 4/5 chick embryos were set up in modified New cultures in the medium containing either the L-type Ca channel blocker, diltiazem, or the N-type Ca channel inhibitor, ω-conotoxin. The embryos were incubated for 22-24 h during which time the control embryos developed, beating looping hearts. At the end of incubation, exposure to the L-type channel blockade with diltiazem resulted in an inhibition of cardiomyogenesis in the most posterior, uncommitted, part of the heart fields. N-type channel blockade with ω-conotoxin was less intense. Cells in the most anterior cardiogenic regions that were already committed at time of exposure continued to differentiate. Thus, regulation and maintenance of normal cytosolic Ca levels are necessary for the early steps of cardiomyocyte specification and commitment leading to differentiation.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Corazón/embriología , Miocitos Cardíacos/citología , Células Madre/citología , Células Madre/metabolismo , Compuestos de Anilina , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/metabolismo , Embrión de Pollo , Diltiazem/farmacología , Corazón/crecimiento & desarrollo , Microscopía Confocal , Contracción Miocárdica , Miocitos Cardíacos/fisiología , Xantenos , omega-Conotoxinas/farmacologíaRESUMEN
Connexin 43 knockout (Cx43alpha1KO) mice have conotruncal heart defects that are associated with a reduction in the abundance of cardiac neural crest cells (CNCs) targeted to the heart. In this study, we show CNCs can respond to changing fibronectin matrix density by adjusting their migratory behavior, with directionality increasing and speed decreasing with increasing fibronectin density. However, compared with wild-type CNCs, Cx43alpha1KO CNCs show reduced directionality and speed, while CNCs overexpressing Cx43alpha1 from the CMV43 transgenic mice show increased directionality and speed. Altered integrin signaling was indicated by changes in the distribution of vinculin containing focal contacts, and altered temporal response of Cx43alpha1KO and CMV43 CNCs to beta1 integrin function blocking antibody treatment. High resolution motion analysis showed Cx43alpha1KO CNCs have increased cell protrusive activity accompanied by the loss of polarized cell movement. They exhibited an unusual polygonal arrangement of actin stress fibers that indicated a profound change in cytoskeletal organization. Semaphorin 3A, a chemorepellent known to inhibit integrin activation, was found to inhibit CNC motility, but in the Cx43alpha1KO and CMV43 CNCs, cell processes failed to retract with semaphorin 3A treatment. Immunohistochemical and biochemical analyses suggested close interactions between Cx43alpha1, vinculin and other actin-binding proteins. However, dye coupling analysis showed no correlation between gap junction communication level and fibronectin plating density. Overall, these findings indicate Cx43alpha1 may have a novel function in mediating crosstalk with cell signaling pathways that regulate polarized cell movement essential for the directional migration of CNCs.
Asunto(s)
Movimiento Celular/fisiología , Conexina 43/genética , Cresta Neural/citología , Actinas/metabolismo , Animales , Western Blotting , Adhesión Celular/genética , Adhesión Celular/fisiología , Movimiento Celular/genética , Polaridad Celular/genética , Polaridad Celular/fisiología , Células Cultivadas , Conexina 43/metabolismo , Conexina 43/fisiología , Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Uniones Comunicantes/metabolismo , Inmunohistoquímica , Inmunoprecipitación , Integrina beta1/metabolismo , Ratones , Ratones Noqueados , Cresta Neural/metabolismo , Cresta Neural/fisiología , Unión Proteica , Vinculina/metabolismoRESUMEN
Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality-associated cardiac anomalies. In animal models, a common read-out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [2002] Dev. Biol. 246:407-417). To address this question further we used a reported loss-of-function approach to interfere with chick CFC expression (Schlange et al. [2001] Dev. Biol. 234:376-389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping.